Throughout this application, certain publications are referenced. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention relates.
According to current immune surveillance theory, the immune system continuously locates and destroys transformed cells. However, some cells escape from an apparently effective immune response and consequently become tumors (1-4). Tumor evasion from immune response is a well established phenomenon demonstrated in numerous studies and is caused by a wide variety of suggested mechanisms (1-4). Among these mechanisms are: the production of suppressive cytokines, the loss of immunodominant peptides, the resistance to killing mechanisms (apoptosis), and the loss of MHC class I (1-4). One of the evasion mechanisms shown to be strongly correlated with tumor progression is the loss or down regulation of MHC class I molecules. This evasion mechanism is abundant in many tumors and can result from a number of different mutations. Several studies revealed weak spots in the MHC class I loading and presentation route including loss of beta-2-microglobulin, TAP1/TAP2 mutations, LMP mutations, loss of heterozygocity in the MHC genes, and down regulation of specific MHC alleles.
Current cancer immunotherapy strategies typically employ the two arms of the immune system: the humoral and the cellular systems. In the first, systemic injection of high affinity monoclonal antibodies (mAbs) directed against cell surface tumor associated antigens has demonstrated statistically significant anti-tumor activity in clinical trials (5,6). Furthermore, anti-tumor mAbs that carry effectors such as cytokines or toxins are currently being evaluated in clinical trials (7). The second major approach for specific cancer immunotherapy employs the cellular arm of the immune system, mainly the CD8+ cytotoxic T-lymphocytes. Two major strategies are currently being used to increase the anti-tumor effectiveness of the cellular arm of the immune system: (i) active immunization of patients with peptides known to be recognized by T-lymphocytes, and (ii) adoptive transfer therapies that enable the selection, activation, and expansion of highly reactive T-cell subpopulations with improved anti-tumor potency. In the first approach, MHC-restricted peptides derived from recently identified tumor associated antigens (such as gp100, the MAGE group, NY-ESO-1) are used to vaccinate patients. These tumor specific antigen-derived peptides are highly specific due to their exclusive expression in specific tissues (8-11). The second strategy, adoptive cell transfer, has recently shown impressive results in metastatic melanoma patients in which highly selected, tumor-reactive T-cells against different over-expressed self-derived differentiation antigens were isolated, expended ex-vivo and reintroduced to the patients. In this approach, a persistent clonal repopulation of T-cells, proliferation in vivo, functional activity, and trafficking to tumor sites were demonstrated (12-14).
A new immunotherapeutic approach recently presented takes advantage of two well-established areas: (i) the known effectiveness of CD8+ cytotoxic T-lymphocytes in the elimination of cells presenting highly immunogenic MHC/peptide complexes, and (ii) the tumor-specific cell surface antigens targeting via recombinant fragments of antibodies, mainly single chain Fv fragments (scFvs). This approach utilizes a recombinant fusion protein composed of two functionally distinct entities: (i) a single-chain MHC class I molecule that carries a highly immunogenic tumor or viral-derived peptide, and (ii) a tumor-specific, high-affinity scFv fragment (15). Several groups have previously shown that a biotinylated MHC peptide multimerized on streptavidin or monomeric HLA-A2/influenza (Flu) matrix peptide complexes coupled via chemical conjugation to tumor-specific antibodies could induce in vitro T-lymphocyte-mediated lysis of coated tumor cells (16-20). However, these approaches utilize chemical conjugation and use whole antibodies or larger fragments, e.g. Fab fragments. However, production and homogeneity owing to the coupling strategy as well as tumor penetration capability are limited due to the large size of such molecules. Lev et al. describe a genetic fusion created between a single-chain recombinant HLA-A2 and tumor specific scFvs. These fusions were shown to be functional in vitro and in vivo, being able to specifically induce T-lymphocyte mediated in vitro and in vivo lysis of target-coated tumor cells (15). The stability of the new chimeric molecule is highly dependent on the presence of the peptide in the MHC groove. Therefore, dissociation of the peptide from the scHLA-A2 domain of the chimeric molecule can impair its stability. Oved et al. addressed this problem by constructing new chimeric molecules in which the peptide is connected to the scHLA-A2/scFv construct via a short linker. This new fusion protein was tested for its in vitro biochemical and biological activity (21).
There is a widely recognized need for a new fusion protein that can maintain its dual activity: bind tumor target cells through the scFv moiety as well as mediate potent, effective and specific cytotoxicity through the recruitment of CD8+ T-cells whose specificity is governed by the covalently linked HLA-A2-restricted peptide.
The MHC class I-restricted CD8+ cytotoxic T-cell (CTL) effector arm of the adaptive immune response is best equipped to recognize tumor cells as foreign and initiate the cascade of events resulting in tumor destruction. However, tumors have developed sophisticated strategies to escape immune effector mechanisms, of which the best-studied is the downregulation of MHC class I molecules which present the antigens recognized by CTLs.
To overcome the limitation of previous approaches and develop new approaches for immunotherapy, a recombinant molecule was constructed in which a single-chain MHC is specifically targeted to tumor cells through its fusion to cancer specific-recombinant antibody fragments or a ligand that binds to receptors expressed by tumor cells.